Glutamate is the most abundant excitatory neurotransmitter in the mammalian central nervous system (CNS) and mediates the fast and slow neurotransmission responsible for such normal neurophysiological processes as memory acquisition and processing, and synaptic plasticity. Postmortem and pharmacological findings strongly implicate dysregulation of glutamate neurotransmission in the pathophysiology of several neuropsychiatric disorders including schizophrenia, Alzheimer disease, Parkinson disease, Huntington disease, epilepsy, attention-deficit hyperactivity disorder, AIDS-related dementia, neuropathic pain, depression, mild cognitive impairment, learning and memory disorders, and others (Lehohla, et al., Metab Brain Dis, 2004; Coyle, et al., Ann. NY Acad. Sci., 2003; Coyle, et al., Curr. Drug Targets CNS Neurol. Disord., 2002; Krystal, et al., Arch Gen Psychiatry, 2002; Dingledine et al., Pharmacol. Rev., 1999; and Ozawa, et al., Prog. Neurobiol., 1998).
Glutamate neurotransmission is mediated by three ionotropic glutamate receptors. These receptors are cation-specific ion channels which regulate fast synaptic neurotransmission. The ionotropic glutamate receptors have been classified into three types: the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, the kainic acid (KA) receptors, and the N-methyl-D-aspartate (NMDA) receptors based on their unique pharmacological, electrophysiological and biochemical properties (Nakanishi, Science, 1992). Furthermore, each of these ionotropic glutamate receptors is made up of multiple heteromeric subunits, contributing to receptor heterogeneity in different tissues (Ozawa, et al., Prog Neurobiol, 1998). However, each of the ionotropic glutamate receptors contain predominant subunits, some requisite for functionality and thought to be most responsible for the regulation of function.
Regulation of the ionotropic glutamate receptors is partly achieved through phosphorylation of specific tyrosine, threonine and serine residues by several kinases and, conversely, through de-phosphorylation of those residues by specific phosphatases (Carvalho, et al., Neurochem. Res., 2000 and Swope, et al., Adv Second Messenger Phosphoprotein Res. 1999). The phosphorylation state of receptor subunits plays a critical role in receptor activity. For example, NMDA receptors are regulated by several kinases and phosphatases acting on its NR1 subunit. Protein kinase C (PKC), and cAMP-dependent protein kinase (PKA) have been shown to phosphorylate serine residues 896 and 897 of the NR1 subunit, respectively (Tingley, et al., J. Biol. Chem., 1997 and Snyder, et al., Neuropharmacology, 2003). Likewise, AMPA receptors are regulated by several kinases and phosphatases acting on the GluR1 subunit; PKA phosphorylates serine residue 845 (Roche, et al., Neuron 16: 1179-1188, 1999; Wang, et al., Science 253: 1132-1135, 1991). Protein phosphatase I (PP1) dephosphorylates these serine residues, thus leading to a molecular switch for receptor activity.
Spinophilin (also named Neurabin II) is a scaffold protein, which is enriched in the dendritic spines of CNS neurons that serve as the major site of glutamatergic synapses in the brain (Allen, et al., Proc. Natl. Acad. Sci. USA, 1997; Hsieh-Wilson, et al., Biochemistry, 1999). Spinophilin was originally identified based on its ability to bind F-actin and protein phosphatase I (PP1). The interaction of spinophilin with PP1 is especially important for the function of ionotropic glutamate receptors as spinophilin acts as a modulator of glutamatergic synaptic neurotransmission by regulating PP1's ability to dephosphorylate the ionotropic glutamate receptors via localization. Evidence for such a function has been demonstrated using voltage whole-cell recordings of kainic acid-induced rundown of AMPA currents in individual acutely dissociated prefrontal cortical neurons (Yan, et al., Nature Neurosci. 1999). In these experiments, agonist-induced rundown of kainic-acid-evoked currents was inhibited by a peptide corresponding to the PP1 binding domain of spinophilin, but not by the same peptide containing a point mutation, thus indicating that when spinophilin no longer interacts with PP1, AMPA receptors (in this example) are no longer dephosphorylated to reduce function; therefore, they remain more active.
In order to discover small molecule compounds that would mimic the action of the spinophilin peptide described above, a novel protein interaction assay between PP1 and spinophilin was utilized to discover inhibitors of binding. These compounds were then evaluated in a whole-cell voltage clamp assay for the ability to inhibit the agonist-induced rundown of AMPA currents and for modulation of NMDA-evoked currents.
Thus, compounds discovered here should have utility in the treatment of several neurospychiatric disorders which have been linked to the dysfunction of glutamate neurotransmission.